Hearing and vestibular function depend on precise variations in stereocilium dimensions according to position in the hair bundle and hair cell type and location in the cochlea or vestibular system. At the core of the stereocilium is a specialized cytoskeletal element, the parallel actin bundle. It displays the hallmarks of a supramolecular scaffold that determines the dimensions, placement and physical properties of stereocilia. Parallel actin bundles are held together by actin-bundling proteins, which cross-link neighboring actin filaments. We discovered and are characterizing a family of novel actin-bundling proteins called the espins. We showed that espin proteins are present throughout the body of stereocilia and the target of the jerker mutation, which causes deafness and vestibular dysfunction in mice. Our scanning electron microscopic analysis of jerker homozygotes, which lack espin proteins, showed that espins are required for stereocilia to undergo a 2-fold increase in diameter during morphogenesis. This led us to hypothesize that espins are required to assemble additional layers of actin filaments at periphery of the pre-existing actin bundle. Espins are encoded by a single gene, but are produced in different sized isoforms (28-91 kDa) with different amino-terminal extensions. We showed that the 116-residue carboxy-terminal actin-bundling module of espins is necessary and sufficient for actin bundling, but shows no obvious resemblance to other actin-bundling proteins. We also characterized the xAB, an additional actin filament-binding site present only in the extended amino-termini of the large espin isoforms, espin 1 and espin 2. We determined that the xAB increases the size of actin bundles formed by espins in vitro and inhibits actin fluorescence recovery (treadmilling) in espin-containing microvilli. We also found that the xAB in the espin 1 isoform is autoinhibited, but can be activated by a peptide in myosin III, a motor that is responsible for compartmentalizing espin 1 to the tip of stereocilia This was an indication that espin isoforms have different activities and localizations in stereocilia. To test our hypothesis that the different espin isoforms play distinct roles in stereocilia, we will compare the activities and localizations of the isoforms we have found in stereocilia. We will use isoform-specific polyclonal antibodies to localize the isoforms during the morphogenesis of stereocilia. In addition, we will use confocal microscopy, negative staining and total interference reflection fluorescence microscopy to examine their effects on actin bundling and polymerization in vitro. To test our hypothesis that espin level determines stereocilium actin filament number and diameter, we will use scanning and transmission electron microscopy to characterize stereocilium morphogenesis and actin bundle organization in our CBA/CaJ congenic mice that lack espins (jerker homozygotes) or contain ~1.5-times normal levels of espins owing to stable expression of an espin-BAC transgene. To deduce the molecular mechanism of actin filament cross-linking by the espin actin-bundling module, we will use scanning alanine mutagenesis to map its actin-binding sites and X-ray crystallography to determine its 3-dimensional structure.